CN107988160A - Human breast cancer cell and its primary it is separately cultured and secondary culture method and purposes - Google Patents
Human breast cancer cell and its primary it is separately cultured and secondary culture method and purposes Download PDFInfo
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Abstract
The invention discloses a kind of human breast cancer cell and its primary it is separately cultured and secondary culture method and purposes.The cell is named as human breast cancer cell HBCC/HL 004, and deposit number is CCTCC NO：C201535.The cell line is that gained is prepared from the cancer tissue samples of patient with breast cancer's surgery excision, has typical cellular biology of tumor characteristic, can continuous Long Term Passages, and character keeps stablizing in vitro.The invention also discloses the preparation method of the cell line at the same time.Breast cancer lines provided by the invention have the biological character of clinically primary breast cancer, available for the pathogenesis of breast cancer, invasion and attack and the Study on Molecular Mechanism of transfer, and the pharmacodynamic study of in-vitro screening cancer therapy drug and detection.
Description
Technical field
The invention belongs to cell biology, it is related to a kind of human breast cancer cell and its primary is separately cultured and passes on training
The method of supporting and purposes.
Background technology
Breast cancer is the common malignant tumour of women, and global epidemiology statistics finds that breast cancer is in female malignant
In morbidity and mortality height rank first, at present, the incidence of China's breast cancer also in trend is gradually increasing, has formed influence
Physically and mentally healthy even one of the hazards of threat to life of women.The treatment means of breast cancer are mainly based on operation, auxiliary
The complex treatment of chemotherapy, but since tumour is in itself there is heterogeneity, i.e., same histological type, differentiation degree are identical to swell
Knurl is different to same drug susceptibility, therefore the medicine of resistance to tumour very likely occurs and causes Endodontic failure.
With new adjuvant chemotherapy progress of research, the individual chemotherapeutic treatment new concept of constantly improve is constantly mentioned, with regard to mesh
For preceding clinical treatment research conditions, how application biotechnology with rapid changepl. never-ending changes and improvements is effectively screened using anticarcinogen, prediction
Individual tumors are to the sensitiveness of medicine, so that the clinical curative effect that improves of the auxiliary treatment strategy instruction for formulating individuation becomes medicine
The hot spot of boundary's research.
In terms of breast cancer individualized treatment, preferable Tumor cell susceptibility system is subject to the weight of domestic and international tumour circle
Depending on.Since for technological layer, the susceptibility results of external medicine and the interior curative effect of medicine of primary cell susceptibility system
There is preferable coincidence rate, there is preferable result repeatability and experimental result success evaluation rate is high.Studies have found that surgery excision
Flesh tissue sample can not only be obtained during original cuiture the tumour cell of high gene reserved, but also because flesh tissue
Just depart from human body, its form, vigor and metabolic characteristic with it is internal extremely similar, have not occurred significant change, either carry out medicine
Quick experiment or the research of carcinogenesis mechanism, are all the cellular biology of tumor characteristics that can most react at that time.
The technology of primary tumor cell in vitro culture is limited to all the time, it is difficult to establish from the primary of tumor patient
Tumor models, reason are that such model foundation success rate is low, mesenchyma stroma of tumors cell mixes, can not long-term cultivation etc..I.e.
Make model foundation success, also have many primary tumor cells can with the change of incubation time or culture environment and occurrence features change
Become, or even lose original characteristic completely.Therefore need constantly to carry out the research of tumour cell primary culture in vitro, continue to optimize culture
Method, propagation and the research that grows and method of the exploitation beneficial to primary cell.
Contain significant quantities of fat tissue in human mammary so that separation and purifying human breast carcinoma primary cell have very big be stranded
It is difficult.But so far, the country more rarely has the report that the breast cancer cell line using Chinese ethnic group as source is established so as to China
The breast cancer research of ethnic group has lacked good experiment material.The present invention it is comprehensive both at home and abroad for breast cancer original cuiture process,
Method and related condition of culture are studied, there is provided a kind of effective human breast carcinoma primary culture method, to be breast cancer
Research and clinical treatment more help is provided.
The content of the invention
The shortcomings that primary and foremost purpose of the present invention is to overcome the prior art and deficiency, there is provided a kind of human breast cancer cell.Should
Cell is separately cultured the breast cancer tissue from clinical patient, which does not import any foreign gene, karyotype 46, X,
X；Genotyping is accredited as a kind of human breast cancer cell line never registered both at home and abroad.
Another object of the present invention is to provide the primary isolated culture method of above-mentioned human breast cancer cell.
It is still another object of the present invention to provide the secondary culture method of above-mentioned human breast cancer cell.
The present invention also aims to provide the purposes of above-mentioned human breast cancer cell.
The purpose of the present invention is achieved through the following technical solutions：
A kind of human breast cancer cell, Classification And Nomenclature are human breast cancer cell HBCC/HL-004, are preserved in Chinese Typical Representative culture
Thing collection, deposit number are CCTCC NO：C201535.
The cell derived is in human breast cancer cell, karyotype 46, X, X；Short tandem repeat (STR) genotype
Represented with 22 tandem repeat locis/allele length：AMEL/X,D3S1358/15/16,D13S317/8/
9,D7S820/8/11,D16S539/9/12,Penta E/11/13,
D2S441/10,TPOX/8/11,TH01/6/9.3D2S1338/22/23/28,CSF1PO/11/12,Penta D/
12/13, D10S1248/13/16, D19S433/15/16, vWA/18/19, D21S11/30, D18S51/17/18, D6S1043/
17/19, D8S1179/11/13, D5S818/7/11, D12S391/18/21, FGA/22/24.
The condition of culture of the human breast cancer cell is preferably to be based on 37 DEG C, 5%CO with HL cultures2Culture；Described
HL culture mediums are：The mixed culture medium of DMEM and Ham ' s F-12NUTRIENT MIX, volume proportion 3:1, while add 5%
Hyclone, and 0.4 μ g/mL cortisols (hydrocortisone), 5 μ g/mL insulin (insulin), 8.4ng/mL
Cholera toxin (cholera toxin), 10ng/mL epidermal growth factor (epithelial growth factor (EGF)), 24
μ g/mL adenines (adenine), 100U/mL penicillin (penicillin) and 100 μ g/mL streptomysins (streptomycin),
0.25 μ g/mL amphotericin Bs (Fungizone), 30 μM of Fasudils (Fasudil) are (referring to the disclosed patent application of inventor
CN103451148A)。
The primary isolated culture method of above-mentioned human breast cancer cell, includes the following steps：
(1) in the case of patient or patient care people's informed consent, the cancerous tissue of patient with breast cancer's surgery excision is collected
Sample.
(2) separated tissue sample is washed with the ethanol of 95~100% (v/v), then is washed with PBS (0.01M, pH7.4), then
Tissue sample is put into the sterile petri dish of the PBS containing precooling, under disecting microscope, tissue is removed with dissection tweezers and scissors
Remaining fat in sample.
(3) tissue sample is digested with digestive juice；Preferably, the digestive juice is to be trained containing the HL of clostridiopetidase A and dispase
Support base.
(4) supernatant is removed in postdigestive tissue centrifugation, and cell precipitation is resuspended in 0.25% (mass volume ratio) pancreas enzyme -EDTA
Middle digestion.
(5) the DMEM culture mediums containing 10% (v/v) FBS are added, supernatant is removed in centrifugation.
(6) dispase and DNase I of tepidarium are added, sample is blown and beaten repeatedly with pipette tips.
(7) the DMEM culture mediums containing 10% (v/v) FBS are added, are hanged with the filter filtration cell in 40~70 μm of apertures
Liquid, collects the cell suspension after filtering, and supernatant is removed in centrifugation.
(8) cell precipitation is resuspended in HL culture mediums, is inoculated in blake bottle culture, obtains human breast cancer cell.
Precooling described in step (2) is preferably in precooling on ice.
The dosage of digestive juice described in step (3) is preferably 10 times of tissue sample volumes.
When the condition of digestion described in step (3) is preferably that 37 DEG C of digestion 1~3 are small.
It is 0.2mg/mL that the concentration of clostridiopetidase A and dispase described in step (3), which is preferably,.
Digestion described in step (4) is when preferably digestion 1 is small on ice or room temperature digests 10 minutes.
Tepidarium described in step (6) is preferably 37 DEG C of tepidarium.
Step (4), (5), the centrifugation described in (7) are preferably that 1000rpm is centrifuged 5 minutes.
The condition of culture described in step (8) is preferably 37 DEG C, 5%CO2。
The secondary culture method of above-mentioned human breast cancer cell, includes the following steps：
(1) when human breast cancer cell is bred to 70~90% abundance, cell is washed with 1 × PBS (0.01M, pH7.4),
Again cell monolayer is digested with 0.05% (mass volume ratio) pancreas enzyme -EDTA.
(2) add DMEM in and digestion reaction；Supernatant is removed in centrifugation, and cell precipitation is resuspended with HL culture mediums, is inoculated in culture
Bottle culture.
The time of digestion described in step (1) is preferably 2~5 minutes.
Centrifugation described in step (2) is preferably that 1000rpm is centrifuged 5 minutes.
The condition of culture described in step (2) is preferably 37 DEG C, 5%CO2。
Above-mentioned human breast cancer cell can be used for pathogenesis, invasion and attack and the Study on Molecular Mechanism of transfer of breast cancer, and
Screen pharmacodynamic study and the detection of cancer therapy drug.
The present invention is had the following advantages that relative to the prior art and effect：
(1) human breast cancer cell provided by the invention, primary to be separately cultured from human breast carcinoma tissue, which, which does not import, appoints
What foreign gene, identifies that karyotype is 46, X, X through karyotyping.
(2) human breast cancer cell provided by the invention, it is primary to be separately cultured from human breast carcinoma tissue, through str locus parting
Identification, is a kind of human breast cancer cell line never registered both at home and abroad.
(3) human breast cancer cell provided by the invention, available for the pathogenesis of breast cancer, invasion and attack and the molecule of transfer machine
System research, and the pharmacodynamic study of in-vitro screening cancer therapy drug and detection.
Brief description of the drawings
Fig. 1 is the cellular morphology figure of human breast cancer cell
The propagation multiplication curve map of Fig. 2 human breast cancer cells
Fig. 3 is the str locus parting figure of human breast cancer cell
Fig. 4 is growthform figure of the human breast cancer cell in 3D differential mediums
Fig. 5 is that human breast cancer cell forms cell clone result figure in soft agar
Fig. 6 is human breast cancer cell heteroplastic transplantation knurl mouse model figure
Fig. 7 is the tissue section strain figure of human breast cancer cell heteroplastic transplantation knurl
Fig. 8 is the tissue ER Immunohistochemical detection result figures of human breast cancer cell heteroplastic transplantation knurl
Fig. 9 is the susceptibility testing result figure of human breast cancer cell
Embodiment
By combination attached drawing described further below it will be further appreciated that the features and advantages of the invention.The implementation provided
Example is only explanation to the method for the present invention, remaining content without limiting the invention in any way announcement.【Embodiment 1】Human milk
The primary of adenocarcinoma cell is separately cultured
(1) in the case of patient or patient care people's informed consent, the breast cancer of patient with breast cancer's surgery excision is collected
Organize 1-2cm3(cubic centimetre), the clinical stages of the patient is T3N0M0, no other to control with 4-9 axillary lymphatic metastasis
Treat.
(2) preparation of digestive juice：HL culture mediums containing clostridiopetidase A and the equal 0.2mg/mL of dispase；Wherein, HL culture mediums are：
The mixed culture medium of DMEM and Ham ' s F-12NUTRIENT MIX, volume proportion 3:1, while 5% hyclone is added,
And 0.4 μ g/mL cortisol (hydrocortisone), 5 μ g/mL insulin (insulin), 8.4ng/mL cholera toxins
(cholera toxin), 10ng/mL epidermal growth factor (epithelial growth factor (EGF)), 24 μ g/mL glands
Purine (adenine), 100U/mL penicillin (penicillin) and 100 μ g/mL streptomysins (streptomycin), 0.25 μ g/
ML amphotericin Bs (Fungizone), 30 μM of Fasudils (Fasudil).
(3) wash separated tissue sample 1 time with the ethanol of 95~100% (v/v), then 2 are washed with PBS (0.01M, pH7.4)
It is secondary, then tissue is put into and is contained in the sterile petri dish of precooling PBS on ice, under disecting microscope, with dissection tweezers and scissors
Remove remaining fat in tissue.
(4) by 1~2mm of tissue sample3(cubic millimeter) is put into 14mL the or 50mL centrifuge tubes of 10mL digestive juices, 37 DEG C
Digest 1 it is small when.
(5) organize low-speed centrifugal (1000rpm) 5 minutes by postdigestive, remove supernatant.
(6) cell precipitation is resuspended in 0.25% (mass volume ratio) pancreas enzyme -EDTA of 2~5mL, be placed in 1 small on ice
When or room temperature 10 minutes.
(7) and then the DMEM culture mediums that 10mL contains 10% (v/v) FBS are added, low speed 1000rmp is centrifuged 5 minutes；As far as possible will
Supernatant removes clean.
(8) the 5mg/mL dispases of 2mL tepidariums (37 DEG C) and the 1mg/mL DNase I of 200 μ L are added, with sterile
P1000 disposable plastics pipette tips blow and beat sample 1 minute repeatedly.
(9) DMEM that 10mL contains 10% (v/v) FBS is added, with the filter filtration cell suspension in 40~70 μm of apertures, is received
Cell suspension after collection filtering, low speed 1000rmp are centrifuged 5 minutes, remove supernatant.
(10) cell precipitation is resuspended in HL culture mediums, is inoculated in the blake bottle culture of T25 or T75, condition of culture is 37
DEG C, 5%CO2。
Successful primary human breast cancer cell is separately cultured according to the method described above, and the form of micro- Microscopic observation cell is as schemed
1, arrangement is close, cell boundary is clear, three-dimensional sense is strong, the epithelial cell of multiangular.The cell classification is named as that " human breast carcinoma is thin
Born of the same parents HBCC/HL-004 ", is preserved in China typical culture collection center (address on April 15th, 2015：Chinese Wuhan
Wuhan University), deposit number CCTCCNO：C201535
【Embodiment 2】The secondary culture of human breast cancer cell
(1) when the human breast cancer cell cultivated in the blake bottle in T25 is bred to 70~90% abundance, with 1 × PBS
(0.01M, pH7.4) washing cell digests 2~5 points of cell monolayer twice, then with 0.05% (mass volume ratio) pancreas enzyme -EDTA
Clock.
(2) add 3mL terminate liquids in and 1~2 point of kind of digestion reaction.
(3) 1000rmp is centrifuged 5 minutes, removes supernatant, and cell precipitation is resuspended in 7mL HL inoculation of medium cultures.
(4) if necessary can be by 1 × 106Human breast cancer cell is resuspended in cells frozen storing liquid (90% hyclone of 1~2mL
And 10%DMSO, v/v) in, it is stored in spare in liquid nitrogen.
Secondary culture human breast cancer cell according to the method described above, cell Proliferation multiplication curve such as Fig. 2 of culture, continuously
Secondary culture 145 days, more than 50 more than generation, human breast cancer cell of the invention remains to keep vegetative state in vitro culture algebraically
Growing multiplication.
【Embodiment 3】The Genotyping identification of human breast cancer cell
(1) human breast cancer cell (1 × 10 of adherent growth6), wash cell twice with 1 × PBS, 0.05% pancreatin-
EDTA digestion cell monolayers 2~5 minutes, in the complete DMEM of 10mL and digestion reaction.
(2) 10000rpm centrifuges 1 minute, to the greatest extent supernatant, adds 200 μ L buffer solutions GA (cell/tissue extracting genome DNAs
Kit DP304, Tiangeng company), vibrate to thorough and suspend.
(3) 20 μ L Proteinase K solution are added, are mixed.
(4) 200 μ L buffer solutions GB (cell/tissue genome DNA extracting reagent kit DP304, Tiangeng company) are added, fully
It is reverse to mix, 70 DEG C of placement 10min, brief centrifugation.
(5) 200 μ L absolute ethyl alcohols are added, fully vibration mixes 15 seconds, brief centrifugation.
(6) resulting solution and flocculent deposit are all added into an adsorption column (cell/tissue extracting genome DNA reagent
Box DP304, Tiangeng company), 12000rpm is centrifuged 30 seconds, removes waste liquid.
(7) 500 μ L buffer solutions GD (cell/tissue genome DNA extracting reagent kit DP304, Tiangengs are added into adsorption column
Company), 12000rpm is centrifuged 30 seconds, removes waste liquid.
(8) 600 μ L rinsing liquids PW (cell/tissue genome DNA extracting reagent kit DP304, Tiangengs are added into adsorption column
Company), 12000rpm is centrifuged 30 seconds, removes waste liquid.
(9) adsorption column is transferred in another centrifuge tube, 50~200 μ L elution buffers is added dropwise to the middle part of adsorbed film
TE (cell/tissue genome DNA extracting reagent kit DP304, Tiangeng company), room temperature placement 2~5min, 12000rpm (~
13400 × g) centrifuge 2 minutes, the DNA solution of extraction is collected into centrifuge tube.
(10) utilize16HS systems (DC2101, promega company) carry out 22 locus (21 STR
Site and 1 gender site) DNA composite amplifications.
(11) ABI is used3100 type genetic analyzers (1.1 edition datas collect software) carry out amplified fragments
Detection.
(12) useWith PowerTyperTM 16Macro software analysis sample datas, automatic gene is carried out
Parting, STR genotyping results are shown in Fig. 3, detect 22 str locus sites, are represented with " str locus seat/allele length "：
AMEL/X,D3S1358/15/16,D13S317/8/9,D7S820/8/11,D16S539/9/12,Penta E/11/13,
D2S441/10, TPOX/8/11, TH01/6/9.3, D2S1338/22/23/28, CSF1PO/11/12, Penta D/12/13,
D10S1248/13/16, D19S433/15/16, vWA/18/19, D21S11/30, D18S51/17/18, D6S1043/17/19,
D8S1179/11/13, D5S818/7/11, D12S391/18/21, FGA/22/24.
The human breast cancer cell of the present invention is a kind of people's mammary gland never registered both at home and abroad through str locus parting
Cancerous cell line.
【Embodiment 4】3D cultures, the fixed and DAPI dyeing of human breast cancer cell
(1) by matrigel (BD, BD Biosciences) in 4 DEG C of dissolvings overnight.
(2) in 8 hole chamber slide of abundant precooling on ice, it is uniformly added into the matrigel tilings bottom of 80 μ L
Into one layer, 37 DEG C of incubator is placed in 15-30 minutes, matrigel is formed glue.
(3) pancreas enzyme -EDTA conventional digestion human breast cancer cell 1-3 minutes, in complete DMEM and digestion reaction.
(4) 1000rmp is centrifuged 5 minutes, removes supernatant, and cell precipitation is resuspended in the HL culture mediums of 150 μ L, adds above-mentioned
(2) in the 8 hole chamber slide prepared, it is placed in 37 DEG C of incubator 15-30 minutes.
(5) upper strata culture medium is washed off.
(6) 5% matrigel is added in the 150 μ L of HL culture mediums of abundant precooling on ice, edge training after micro pipette tips mix
Foster wooden partition is carefully added into 8 hole chamber slide.
(7) in 37 DEG C, 5%CO2Under the conditions of cultivate 7 days, replace a subculture within every 2 days.
(8) dyed and in fluorescence microscopy Microscopic observation with DAPI.
The human breast cancer cell cultivated according to the method described above in 3D differential mediums, after fixed and DAPI dyeing, shows
(cell is the cell product group of non-normal differentiation to form such as Fig. 4 of micro- Microscopic observation cell, does not form close clearly knot of tissue
Structure)
【Embodiment 5】The soft-agar cloning of human breast cancer cell forms experiment
(1) the low melting-point agarose liquid of 1.2% and 0.8% two concentration is prepared respectively with distilled water, after autoclaving, dimension
Hold and ensure that it is not solidified in 40 DEG C of water-bath.
(2) according to 1:1 ratio make 1.2% agarose and 2 × DMEM or 2 × HL culture mediums (containing 2 × antibiotic and
20% calf serum) mixed in sterile centrifugation tube, take 3mL mixed liquors to inject in 6 orifice plates, cooled and solidified, as bottom fine jade
Fat puts 37 DEG C, and spare in 5%CO2 incubators, every plant of cell does three multiple holes.
(3) according still further to 1:1 ratio make 0.8% agarose and 2 × DMEM or 2 × HL culture mediums in sterile centrifugation tube
Mix, then the cell suspension of addition 2mL (adds the agar containing HeLa cells into pipe in agarose and 2 × DMEM mixed liquors
The cell suspension containing human breast cancer cell is added in sugared cell suspension, agarose and 2 × HL culture mediums), per hole in six orifice plates
Add 3 × 104A cell, fully mixes, and injection is covered with 1.2% agarose bottom plate, forms double agar layers.Treat top-layer agar
After solidification, 37 DEG C are inserted, is cultivated 30 days in 5%CO2 incubators.
(4) plate is placed under inverted microscope, observes the form of cell clone and the oncogenicity of cell.
HeLa cells and human breast cancer cell such as Fig. 5 of the growth conditions in soft agar, HeLa cells can be in soft agar
Significantly greater cell clone is formed, human breast cancer cell also forms cell clone in agar.Illustrate that human breast cancer cell has
There is the ability of grappling-dependent/non-dependent growth, increment is abnormal in vitro, has oncogenicity.【Embodiment 6】Human breast cancer cell allosome
Transplantable tumor mouse model
(1) it is in human breast cancer cell (the cell count about 1 × 10 of exponential phase6), pancreas enzyme -EDTA conventional digestion 3-
5 minutes, in complete DMEM and digestion reaction.
(2) 1000rmp is centrifuged 5 minutes, removes supernatant, and cell precipitation is resuspended in Matrigel HC (BD, the BD of 200 μ L
Biosciences in).
(3) 6 week old male mices of severe combined immunodeficient, by the Matrigel HC suspensions of human breast cancer cell, warp
Subcutaneous vaccination mouse, left and right 2 sites (see Fig. 6) of every mouse inoculation, amount to 10 sites (5).
(4) size such as Fig. 6 of transplantable tumor is observed and measured weekly, and mouse is put to death after 8 weeks.
(5) transplantable tumor for taking mouse to be formed, conventional organization embedding, section, HE dyeing, micro- Microscopic observation are simultaneously photographed.
The human breast cancer cell heteroplastic transplantation knurl established according to the method described above, after organization embedding, fixation and HE dyeing, shows
Pathological section such as Fig. 7 of micro- Microscopic observation breast cancer tissue (most cell colors are deep, growth of tumour cell molecular marker for increased proliferation).
Patient's tumor tissues and the transplantable tumor of mouse formation, conventional organization embedding, section, DAB dyeing, microscope are taken at the same time
Lower observation is simultaneously photographed.Coloration result such as Fig. 8, the nucleus of the visible positive brown color of two tumor tissues, i.e., express female
Hormone receptor
【Embodiment 7】The chemotherapy drug susceptibility detection of human breast cancer cell
(1) 0.05% pancreatin of human breast cancer cell is digested, is prepared into single cell suspension, be inoculated in 96 orifice plates, per hole
100 μ L of inoculating cell suspension, are about 5000 cells per hole, in 37 DEG C, 5%CO2Incubator culture.
(2) 5 it is small when after dosing (taxol, adriamycin) handle, per hole add the prepared various concentrations medicines of 200 μ L,
Taxol drug concentration gradient (μM) is 0.1,0.3,1,3,10,30,100, adriamycin drug concentration gradient (μM) for 0.0016,
0.008th, 0.04,0.2,1,5,25, each gradient of every kind of medicine sets six multiple holes, while sets the (inoculation of cell controls group
Cell not agent-feeding treatment) and only plus HL culture mediums blank control group, six multiple holes of every group of setting.Taxol be used for oophoroma and
A line and second line treatment for breast cancer and NSCLC；Adriamycin is antimitotic and cytotoxic drug, can successfully be induced more
The alleviation of kind malignant tumour, including acute leukemia, lymthoma, soft tissue and osteosarcoma, children malignant tumors and adult solid
Knurl, particularly for breast cancer and lung cancer.
(3) drug-treated (37 DEG C, 5%CO2Incubator culture) 48 it is small when after, suck solution in hole, per hole add 10 μ
LCKK-8 detection reagents (colleague's chemistry, Japan), i.e. 10 μ LCCK-8+90 μ LDMEM culture mediums.
(4) in 37 DEG C, 5%CO2Continue in cell incubator 1~1.5 hour of incubation, range of absorbency is controlled 1.0
It is best between~1.5.
(5) absorbance at 450nm is measured with microplate reader.
The sensitivity Detection result such as Fig. 9 of human breast cancer cell to above-mentioned 2 kinds of anti-cancer medicine paclitaxels, adriamycin.Japanese yew
To cell survival rate 80% when alcohol is below 0.1 μM of low concentration, when 3 μM of high concentration, makes cell survival rate be reduced to 40%.And Ah
Mycin low concentration just show it is obvious kills cytosis, 0.2 μM can make cell survival rate be down to 50%, during 5 μM of high concentration then
It is only 10% to make cell survival rate.Experiment shows sensitiveness and discrimination of the human breast cancer cell to different cancer therapy drugs above.
Above-described embodiment is the preferable embodiment of the present invention, but embodiments of the present invention and from above-described embodiment
Limitation, other any Spirit Essences without departing from the present invention with made under principle change, modification, replacement, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
1. a kind of human breast cancer cell, it is characterised in that be named as human breast cancer cell HBCC/HL-004, deposit number is
CCTCC NO：C201535.
2. human breast cancer cell according to claim 1, it is characterised in that chromosome 46, X, X, Short tandem repeatSTR sequence
Row genotype is represented with 22 tandem repeat locis/allele length：AMEL/X,D3S1358/15/16,
D13S317/8/9,D7S820/8/11,D16S539/9/12,Penta E/11/13,D2S441/10,TPOX/8/11,TH01/
6/9.3, D2S1338/22/23/28, CSF1PO/11/12, PentaD/12/13, D10S1248/13/16, D19S433/15/
16, vWA/18/19, D21S11/30, D18S51/17/18, D6S1043/17/19, D8S1179/11/13, D5S818/7/11,
D12S391/18/21,FGA/22/24。
3. the primary isolated culture method of the human breast cancer cell described in claim 1 or 2, it is characterised in that including following step
Suddenly：
(1) in the case of patient or patient care people's informed consent, the cancer tissue samples of patient with breast cancer's surgery excision are collected；
(2) separated tissue sample is washed with the ethanol of 95~100% (v/v), then is washed with PBS (0.01M, pH7.4), then by group
Tissue samples are put into the sterile petri dish of the PBS containing precooling, and under disecting microscope, tissue sample is removed with dissection tweezers and scissors
In remaining fat；
(3) tissue sample is digested with digestive juice；Preferably, the digestive juice is to be cultivated containing the HL of clostridiopetidase A and dispase
Base；
(4) supernatant is removed in postdigestive tissue centrifugation, and cell precipitation is resuspended in 0.25% (mass volume ratio) pancreas enzyme -EDTA and is disappeared
Change；
(5) the DMEM culture mediums containing 10% (v/v) FBS are added, supernatant is removed in centrifugation；
(6) dispase and DNase I of tepidarium are added, sample is blown and beaten repeatedly with pipette tips；
(7) the DMEM culture mediums containing 10% (v/v) FBS are added, with the filter filtration cell suspension in 40~70 μm of apertures, are received
Supernatant is removed in cell suspension after collection filtering, centrifugation；
(8) cell precipitation is resuspended in HL culture mediums, is inoculated in blake bottle culture, obtains human breast cancer cell.
4. the primary isolated culture method of human breast cancer cell according to claim 3, it is characterised in that：
Precooling described in step (2) is in precooling on ice；
The dosage of digestive juice described in step (3) is 10 times of tissue sample volumes, and the condition of the digestion disappears for 37 DEG C
When change 1~3 is small；
The concentration of clostridiopetidase A and dispase described in step (3) is 0.2mg/mL.
5. the primary isolated culture method of human breast cancer cell according to claim 3, it is characterised in that：
Digestion described in step (4) is when digestion 1 is small on ice or room temperature digests 10 minutes；
The tepidarium that tepidarium described in step (6) is 37 DEG C；
The condition of culture described in step (8) is 37 DEG C, 5%CO2。
6. the primary isolated culture method of human breast cancer cell according to claim 3, it is characterised in that：
Step (4), (5), the centrifugation described in (7) centrifuge 5 minutes for 1000rpm.
7. the secondary culture method of the human breast cancer cell described in claim 1 or 2, it is characterised in that include the following steps：
(1) when human breast cancer cell is bred to 70~90% abundance, cell is washed with PBS, then disappeared with 0.05% pancreas enzyme -EDTA
Change cell monolayer；
(2) add DMEM in and digestion reaction；Supernatant is removed in centrifugation, and cell precipitation is resuspended with HL culture mediums, is inoculated in blake bottle training
Support.
8. the secondary culture method of human breast cancer cell according to claim 7, it is characterised in that：
The time of digestion described in step (1) is 2~5 minutes；
Centrifugation described in step (2) centrifuges 5 minutes for 1000rpm, and the condition of the culture is 37 DEG C, 5%CO2。
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| CN115322967B (en) * | 2022-06-13 | 2023-08-08 | 浙江省人民医院 | Immortalized human papillary thyroid carcinoma fibroblast cell line and its construction method and application |
| CN117070462A (en) * | 2023-08-17 | 2023-11-17 | 镜像绮点(上海)细胞技术有限公司 | Isolation and purification of primary cells |
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